It has been recognized for many years that cyanate readily reacts with certain amino acid side chain functional groups of a peptide [see G. R. Stark, Method In Enzymology 11, 590-594 (1967)]. While carbamylation is most rapid at sulfhydryl and imidazole sites, the resulting reaction products are of little concern due to their rapid reversal in slightly alkaline buffers. Modification of peptidyl primary amines (for example, NH.sub.2 -terminus and lysine residues) occurs at an appreciable rate and, for all practical purposes, is irreversible [see G. R. Stark, W. H. Stein, and S. Moore, J. Biol. Chem. 235, 3177-3181 (1960)]. At each site of primary amine carbamylation the peptide is reduced in physiological buffers one positive charge, thereby often resulting in diminished peptidyl solubility and/or biological activity. Since cyanate is an equilibrium product of aqueous urea solutions [see J. R. Marier and D. Rose, Anal. Biochem. 7, 304-314 (1964)], all peptides containing reactive functional groups, when handled in the presence of urea, are susceptible to irreversible carbamylation. Urea, being an excellent peptidyl solvent due to its ability to disaggregate structural order, facilitates carbamylation. These undesirable derivatized forms not only represent immediate losses in yield but also constitute complications in purification processes.
To diminish carbamylation of peptides, urea solutions are freshly freed of cyanate prior to use, and all chemical manipulations are conducted at reduced temperatures. The removal of cyanate by deionization or through pH reduction to below 2.0 is at best temporary, since ammonium cyanate reappears as an equlibrium product of aqueous urea. The low temperature operational restriction results in slower chemical reactions and reduced chromatographic performance. An excellent example of this problem can be seen in the production and purification of insulin. Insulin generally is purified at 4.degree. C. in the presence of aqueous urea, usually 7M in concentration. Aqueous urea is well recognized as a reagent useful in inhibiting insulin polymerization and self-aggregation. However, carbamylated amino products are known to accumulate during purification even when using freshly deionized urea and low temperatures.
The discovery which forms the basis of the present invention resides in the use of a reagent from a class of reagents in the presence of the cyanate-containing or cyanate-generating medium. The properties of this reagent are ideally suited to scavenge cyanate under the customary peptide processing conditions before it can attack and carbamylate the product peptide.